The chemotactic behavior of E. coli provides the best-studied and most tractable system for elucidating the molecular machinery that also underlies more complex bacterial behaviors, such as host invasion and pathogenesis. A detailed understanding of how bacteria detect and follow chemical gradients in their environment may well lead to the development of novel antimicrobial therapies. The objective of this work is to elucidate the mechanisms responsible for signal amplification by bacterial chemoreceptors. Physical clustering of the cell's receptors appears to provide an important source of gain in the system and recent genetic and crosslinking studies of the serine and aspartate receptors indicate that they signal collaboratively in "teams" comprised of trimers of receptor dimers. Moreover, receptor teams can contain chemoreceptors with different detection specificities working together to control shared kinase molecules. To test predictions of the team model of receptor signaling, an in vivo crosslinking test will be used to screen a large existing collection of receptor mutants for defects in trimer-of-dimers formation. Trimer-defective mutants will then be subjected to a battery of in vivo signaling tests to ascertain which receptor functions - stimulus detection, transmembrane signaling, kinase activation, and clustering - depend on the trimer of dimers structural organization. In an independent test of the correlation between receptor function and trimer formation predicted by the team signaling model, pseudorevertants of trimer-defective receptor mutants that have regained the ability to mediate serine chemotaxis will be examined for comcomitant restoration of trimer-forming ability. These studies should provide important new perspectives on the issue of receptor clustering, a hallmark of biological signaling systems at every complexity level. This work will be done primarily at the National University in Mar del Plata, Argentina, in collaboration with Dr. Claudia Studdert as an extension of NIH grant # RO1 GM19559.